There is a fundamental gap in understanding how human articular cartilage derived heparan sulfate proteoglycans bind to and modulate the activity of heparin binding growth factors important for regulating cartilage structural integrity and function. The long term goal is to understand the role of ECM molecules in regulating cartilage health, and to use this knowledge in the treatment and prevention of cartilage disorders. The objective of this particular proposal is to identify a role for the heparan sulfate proteoglycan, perlecan in modulating VEGF-A bio-availability, in human osteoarthritic cartilage. VEGF-A activity contributes to articular cartilage extracellular matrix degradation by differentially regulating matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression and secretion. Heparan sulfate proteoglycans act as co- receptors in the regulation of VEGF-A activity, and perlecan is well positioned within osteoarthritic cartilage to have such a role. The central hypothesis is that perlecan regulates VEGF-A bio-availability in osteoarthritic cartilage by sequestration through heparan sulfate interaction. The rationale for the proposed project is that the identification of heparan sulfate structures on perlecan that regulate VEGF-A bio-availability could have a significant positive impact on future strategies to treat osteoarthritis cartilage degradation. Thus, the proposed research is relevant to that part of NIH's mission that pertains to supporting research investigating the causes, treatment, and prevention of arthritis and musculoskeletal diseases, as well as the training of basic scientists to conduct this research. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify sulfate groups within the glycosaminoglycan chains attached to perlecan important for VEGF-A binding;and 2) Identify the requirements for articular cartilage derived perlecan to bind and modulate VEGF-A bio-availability in vitro. Under the first aim, an already proven cesium chloride density gradient centrifugation and anion exchange chromatography approach will be used to purify perlecan from human osteoarthritic cartilage. HPLC and immunoblotting approaches already will then be employed to characterize the disaccharide composition of perlecan. Under the second aim, solid phase immunoblotting and cell culture based approaches will be employed to determine the requirement for perlecan/VEGF-A interaction and function in vitro. The approach is innovative, because it utilizes human articular cartilage heparan sulfate proteoglycan, derived from healthy and diseased tissues to better understand the complexity of mechanisms that regulate cartilage degradation. The proposed research is significant, because it is expected to advance and expand understanding of how heparan sulfate proteoglycans bind to and modulate the activity of heparin binding growth factors in osteoarthritic cartilage, knowledge needed to develop novel pharmacologic strategies that aim to regulate such factors in slowing or preventing osteoarthritic cartilage degradation.Project Narrative The proposed investigation is of an important and under-investigated area of articular cartilage biology and osteoarthritis that has potential to enhance our understanding of mechanisms that regulate articular cartilage degradation. The proposed research has relevance to public health, because articular cartilage degradation is a primary pathology associated with osteoarthritis. Thus, findings are expected to be applicable to the health of humans.